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Abstract

Background— Accumulation of excess cholesterol by intimal arterial smooth muscle cells (SMCs) contributes to the formation of foam cells in atherosclerotic lesions. The purpose of this study was to examine the expression and activity of ATP-binding cassette transporter A1 (ABCA1) in model intimal and medial arterial SMCs, in human atherosclerotic coronary artery intimal and medial layers, and in human intimal and medial SMCs.

Smooth muscle cells (SMCs) constitute a, or the, major cell type in most stages of atherosclerosis.1 Relatively little is known, however, about cholesterol homeostasis in arterial SMCs compared with macrophages. Like macrophages, intima-type SMCs express scavenger receptors and develop into smooth muscle foam cells containing excess cholesteryl esters.2–6 Removal of cholesterol from SMCs, like other cells, is dependent on the binding of apolipoprotein A-I (apoA-I) to the cell surface and the transfer of cellular phospholipids and cholesterol to apoA-I through the actions of the ATP-binding cassette transporter A1 (ABCA1).7,8 Previous studies have reported marked variability in apoA-I binding and apoA-I–mediated lipid efflux from arterial SMCs obtained from different species,9,10 suggesting that the apoA-I–SMC interaction may differ depending on SMC phenotype. Recent studies have reported both increased and decreased expression of ABCA1 in atherosclerotic arteries,11–13 but the relative expression of ABCA1 in intimal versus medial layers has not been described. Although ABCA1 is expressed in SMCs originating from the medial layer of normal arteries,7 its expression in intimal SMCs has not been reported.

Clinical Perspective on p 3231

In addition to excess cholesterol accumulation, intimal SMCs are characterized by a dedifferentiated state, an increased rate of proliferation, a loss of contractility, an increased synthesis of extracellular matrix components, and a reduced expression of SMC markers, including smooth muscle α-actin and smooth muscle myosin heavy chain.14,15 The marked differences in gene expression patterns have led some investigators to propose that SMCs populating the intima are of different clonal origins than those in the medial layer.14,16,17 Intimal SMCs of human origin are not available for use in continuous culture studies. In the present studies, we used clones of rat SMCs that exhibit features of human intimal SMCs, including accumulation of excess cholesteryl esters and reduction of smooth muscle α-actin and myosin heavy chain, and exhibit an epithelioid or cuboidal morphology as a model for cholesterol mobilization from intimal SMCs. We compared these results with the expression of ABCA1 in the intima and media and in intimal and medial SMCs of atherosclerotic human coronary arteries. Our results indicate a markedly reduced expression of ABCA1 and reduced binding and lipid mobilization by apoA-I from the model intimal SMCs and a reduction of ABCA1 expression in human atherosclerotic intima and intimal SMCs. Overexpression of ABCA1, however, failed to correct apoA-I binding to, or cholesterol efflux from, intima-phenotype SMCs.

Methods

SMC Cultures

Clonal SMC lines obtained from the medial layer of thoracic aortas of 12-day-old Wistar-Kyoto (WKY) rats (WKY12–22, epithelioid morphology) and 3-month-old rats (WKY3M–22, spindle morphology) were a generous gift from Dr Joan Lemire (University of Washington, Seattle).18 Cells were grown in DMEM/FBS and used between the 2nd and 10th passage after reestablishing the culture. Cells maintained a constant growth rate, morphology, ABCA1 expression, and apoA-I responses during these passages. Epithelioid SMCs showed a somewhat lower rate of cell proliferation at confluence (Figure I of the online-only Data Supplement). In some experiments, cells were loaded with excess nonlipoprotein cholesterol to generate SMC foam cells before incubation with apoA-I.10

Lipid Analyses

ApoA-I was obtained from the delipidated whole-protein fraction of human plasma high-density lipoprotein (HDL).10 Radiolabeled lipid efflux studies were performed as described in the Methods section of the online-only Data Supplement.19 Cholesterol mass was determined by gas chromatography.20 Cell surface cholesterol was determined with cholesterol oxidase as previously described.21 The rate of degradation of [14C]cholesteryl esters was assessed in the presence of an acyl CoA:cholesterol acyltransferase inhibitor as previously described.22 Two-dimensional gel electrophoresis of HDL particles was performed as described in the online Methods section.20

ABCA1 Analysis in SMCs

Determination of ABCA1 mRNA levels by quantitative real-time polymerase chain reaction and assessment of total, crude cell membrane, cell surface, and phosphorylated ABCA1 protein were as described in the Methods section in the online-only Data Supplement.

Cell Surface Binding of ApoA-I

Cells were incubated for 2 hours at 4°C in DMEM/BSA containing 25 mmol/L HEPES and 10 μg/mL 125I–apoA-I ±200 μg/mL unlabeled apoA-I. Cells were washed 5 times with cold PBS/BSA and twice with cold PBS before solubilization in 0.2N NaOH for quantification of radioactivity and protein. Cross-linking of apoA-I to ABCA1 was performed as described in the online-only Methods section.

ABCA1 Analysis in Atherosclerotic Human Coronary Arteries

Coronary artery sections from 14 patients with the primary diagnosis of coronary heart disease were obtained from the Cardiovascular Registry at St Paul’s Hospital, University of British Columbia (Table I of the online-only Data Supplement). Oil Red O staining, in situ hybridization of ABCA1 mRNA, immunohistochemistry for smooth muscle α-actin and ABCA1, and image analysis were performed as described in the online-only Data Supplement.

Statistical Analysis

Results for cell culture data are expressed as mean±SD and for in situ hybridization and immunofluorescence data as mean±SEM. Significant differences between experimental groups were determined with the Student t test, with a value of P<0.05 considered significant.

Results

Cell Models

We previously determined that binding of apoA-I and apoA-I–mediated cholesterol and phospholipid efflux was intact in human but not rat SMC lines,10 but we did not correlate these results with features of medial or intimal SMCs in either species. In the absence of human intimal SMCs suitable for use in repeated cell culture studies, we studied clonal SMCs obtained from the aorta of 12-day-old WKY rats with features consistent with human intimal (plaque) SMCs14 and SMCs from bovine and pig arterial intima,24,25 including increased proliferation and proteoglycan production and reduced smooth muscle α-actin and smooth muscle myosin heavy chain expression, that demonstrate an epithelioid morphology in culture.18 We compared these with SMCs obtained from the medial layer of 3-month-old WKY rats exhibiting a spindle morphology as our medial SMC model (Figure 1A).18

Figure 1. Morphology and lipid efflux to apoA-I from model arterial SMCs. A, Phase-contrast photomicrographs of SMC clones derived from aortas of 3-month-old (spindle) or 12-day-old (epithelioid) WKY rats. Bar=100 μm. B, SMCs loaded with cholesterol and radiolabeled with [3H]choline chloride for 24 hours were incubated with the indicated concentration of apoA-I for 24 hours to determine the efflux of phosphatidyl[3H]choline (PC) (i) and [3H]sphingomyelin (SM) (ii). Results are average±SD of 2 experiments performed in quadruplicate and expressed as the percentage of total cellular plus medium [3H]counts for PC or SM in the medium after subtraction of efflux to medium containing 1 mg/mL BSA alone. C, SMCs loaded with cholesterol and equilibrated were incubated for 24 hours in DMEM/BSA with or without 10 μg/mL apoA-I. At the end of the incubation, media and cells were collected and total lipids were extracted. Unesterified cholesterol (UC) mass in the medium (i), cholesteryl ester (CE) mass in SMCs (ii), and UC mass in SMCs (iii) were determined by gas chromatography. Results represent average±SD of 2 experiments performed in quadruplicate.

Removal of cell phospholipids by apoA-I and other HDL apolipoproteins is required for initial HDL formation and to allow the developing HDL to solubilize cholesterol also removed from cells. Spindle-morphology SMCs readily released both phosphatidylcholine and sphingomyelin, whereas epithelioid SMCs released little or none of these phospholipids to apoA-I (Figure 1B). Spindle SMCs similarly showed release of cholesterol mass to apoA-I, whereas epithelioid SMCs showed very little release of cholesterol to albumin alone or to apoA-I (Figure 1Ci). ApoA-I actively depleted cell cholesteryl ester mass from spindle SMCs but induced no reduction of the larger pool of cholesteryl ester mass seen in WKY rat epithelioid SMCs after loading with nonlipoprotein cholesterol (Figure 1Cii). Efflux of radiolabeled cholesterol to the medium and a decrease in radiolabeled cholesteryl esters in cells by apoA-I showed patterns similar to cholesterol mass changes in spindle and epithelioid SMCs (Figure II of the online-only Data Supplement). Additional clonal spindle and epithelioid SMCs obtained from Sprague-Dawley adult and pup rats, as well as spindle-morphology human arterial SMCs and the epithelioid human SMC clone HITA2,17 showed similar presence or absence of efflux of cell cholesterol to apoA-I, respectively (Figure III in the online-only Data Supplement). Neither the Sprague-Dawley rat or human HITA2 epithelioid SMCs, however, accumulated cholesteryl esters the way atherosclerotic and neointimal SMCs do4,5 (Figure III in the online-only Data Supplement). For this reason, the WKY rat epithelioid and spindle SMC lines were chosen for subsequent studies as our models for intimal and medial SMCs, respectively.

Decreased ABCA1 Expression and ApoA-I Binding in Epithelioid SMCs

ApoA-I–mediated phospholipid and cholesterol efflux is critically dependent on the membrane transporter ABCA1, the expression of which is normally increased with increasing cell cholesterol content.8 Consistent with our lipid efflux results, we found low basal and cholesterol-stimulated levels of ABCA1 mRNA and protein in epithelioid-morphology WKY rat SMCs (Figure 2A and 2B). Spindle SMCs showed high basal and further increased ABCA1 expression on cholesterol loading. Similar results were found for human and Sprague-Dawley rat SMCs, with spindle SMCs from both species showing increased ABCA1 protein with cholesterol loading and epithelioid cells showing an absence of increase in ABCA1 protein with cholesterol loading (Figure IV of the online-only Data Supplement).

Figure 2. ABCA1 expression and apoA-I binding by arterial SMCs. A, SMCs were grown to confluence in DMEM/10% FBS, incubated with or without 30 μg/mL cholesterol for 24 hours, and equilibrated in DMEM/BSA for 24 hours before determination of ABCA1 mRNA levels by real-time polymerase chain reaction. ABCA1 mRNA was normalized to cyclophilin mRNA levels, with the ratio of ABCA1 to cyclophilin mRNA levels in non–cholesterol-loaded spindle cells set at 1. Results are average±SD of 3 experiments. B, ABCA1 protein in cells prepared as in A and detected by Western blotting of 50 μg cellular membrane protein with a rabbit polyclonal antibody to ABCA1. Results are representative of 3 experiments with similar results. C, SMCs grown to confluence and loaded with cholesterol as in A were incubated with 10 μg/mL 125I–apoA-I ±200 μg/mL unlabeled apoA-I for 2 hours at 4°C. After extensive rinsing, cells were assessed for radioactivity. Cell surface 125I-apoA bound per 1 mg cell protein was determined by subtracting values in the presence of unlabeled apoA-I. Results are the mean±SD of quadruplicate determinations and are representative of 3 experiments with similar results.

A direct binding interaction between apoA-I and ABCA1 and/or a cellular association of apoA-I with lipid domains formed by ABCA1 are necessary for apoA-I–mediated lipid efflux.26 Spindle-morphology rat SMCs showed a much higher level of apoA-I binding than epithelioid SMCs before cholesterol loading and an approximate doubling of apoA-I binding after loading with nonlipoprotein cholesterol (Figure 2C). Despite no significant increase in ABCA1 expression in epithelioid SMCs after cholesterol loading, there was a small but significant increase in the low level of apoA-I binding to these cells after cholesterol loading (Figure 2C).

To test the relevance of our results using cultured epithelioid and spindle arterial SMCs, we examined the relative expression of ABCA1 mRNA in the intimal and medial layers of coronary artery sections from 14 patients with various degrees of coronary artery disease resulting from native, as opposed to postangioplasty or posttransplantation, atherosclerosis (clinical details in Table I of the online-only Data Supplement). As previously demonstrated for transplant27 but not native human coronary atherosclerosis, staining with Oil Red O and costaining with smooth muscle α-actin showed a marked accumulation of lipid in intimal but minimal lipid accumulation in medial SMCs (Figure 3A and 3B). In situ hybridization revealed a striking decrease in ABCA1 mRNA levels in the intimal compared with medial layers of atherosclerotic human coronary arteries (Figure 3D). ABCA1 mRNA was present in an average of only 26% of total intimal area compared with 49% of total medial artery area from the 14 specimens (Figure 3E).

To test whether human intimal SMCs specifically exhibit diminished ABCA1 expression, we determined the relative level of ABCA1 protein in smooth muscle α-actin–positive cells in medial and intimal layers of coronary artery sections from the same 14 patients. Costaining of smooth muscle α-actin and ABCA1 revealed a relative paucity of ABCA1 protein in SMCs in the proximal intima (Figure 4C, top three fourths of the layer above the internal elastic lamina) relative to the deep intimal and medial layers. Image analysis of total ABCA1 immunoreactivity and colocalization of ABCA1 and smooth muscle α-actin per total smooth muscle α-actin–staining area in intimal or medial layers from 14 patients revealed significantly lower total ABCA1 and SMC-specific ABCA1 in atherosclerotic intima compared with medial arterial layers (Figure 4E and 4F, respectively). These findings are consistent with our results of diminished ABCA1 expression in model intimal rat and human epithelioid SMCs.

Figure 4. ABCA1 and smooth muscle α-actin immunoreactivity in the intima (I) and media (M) of atherosclerotic human coronary arteries. Representative dark-field photomicrographs of smooth muscle α-actin (A; green) and ABCA1 (B; red) detected immunohistochemically. C, Color segmentation analysis showing colocalization of ABCA1 and smooth muscle α-actin (pink). The green line represents the internal elastic lamina. Scale bar in A=50 μm. D, Hematoxylin and eosin–stained adjacent section highlighting the region shown in A through C. Scale bar in D=200 μm. E, ABCA1 immunoreactivity as a percentage of total area is significantly higher in media (53%) compared with intima (31%), with intimal area measured as all area above the internal elastic lamina. F, Colocalization of ABCA1 and smooth muscle α-actin as a percentage of total smooth muscle α-actin–staining area indicates a significantly higher degree of ABCA1 immunoreactivity in SMCs in media (43%) vs intima (28%). E and F, Mean±SEM of averages from the 14 cases, with at least 2 fields of view used to calculate an average for each section.

We next determined whether increased ABCA1 expression corrects binding and lipid efflux to apoA-I by epithelioid SMCs. LXR agonist TO901317 treatment increased total and cell surface ABCA1 in both spindle and epithelioid SMCs (Figure 6A). Consistent with this increase, spindle SMCs treated with the LXR agonist showed a >2-fold increase in cholesterol efflux to apoA-I (Figure 6Ba). Epithelioid cells, in contrast, showed no increase in cholesterol efflux to apoA-I despite increased ABCA1 expression, nor did they exhibit any correction of phosphatidylcholine efflux to apoA-I (Figure V of the online-only Data Supplement). The ability of apoA-I to be cross linked directly to ABCA1 increased after LXR agonist treatment of spindle SMCs but was low and increased minimally after agonist treatment of epithelioid SMCs (Figure VI in the online-only Data Supplement). The effects on lipid efflux were mirrored by changes in HDL particle formation as assessed by 2-dimensional gel electrophoresis. Spindle SMCs showed an increase in formation of all subspecies of α-HDL after LXR agonist treatment, whereas epithelioid SMCs showed pre-β but little or no α-HDL formation either before or after treatment with the agonist (Figure 7).

Figure 6. Cell surface localization of ABCA1 and cholesterol efflux from arterial SMCs treated with the LXR agonist TO901317. A, Confluent SMCs treated in the absence or presence of cholesterol and/or 10 μmol/L TO-901317 were biotinylated, and total and cell surface ABCA1 levels were determined by Western blotting as described in Methods. Protein disulfide isomerase (PDI) levels were determined as loading control. Results are representative of 2 experiments with similar results. B, Confluent SMCs labeled with [3H]cholesterol during growth were loaded with cholesterol, equilibrated, and incubated with the indicated concentration of apoA-I for 24 hours. Equilibration of cholesterol-loaded cells and apoA-I treatment were performed in the absence or presence of 10 μmol/L TO-901317. At the end of the incubation period, media were collected, and unesterified cholesterol (UC) in the medium and in cell cholesteryl ester (CE) and UC were analyzed for [3H]cholesterol. Results are expressed as percentage of total cell plus medium [3H]sterol in the medium (a), cell CE (b), and cell UC (c) after subtraction of efflux to medium containing 1 mg/mL BSA alone. Values are the mean±SD of quadruplicate determinations and are representative of 2 experiments with similar results.

Figure 7. HDL particles formed by spindle and epithelioid arterial SMCs. Confluent SMCs were loaded with cholesterol, equilibrated for 24 hours, and incubated with 10 μg/mL apoA-I for 24 hours in the absence or presence of 10 μmol/L TO-901317 (LXR). Cell media were collected and analyzed for apoA-I–containing HDL particle species by 2-dimensional gel electrophoresis. Results are representative of 2 separate experiments with similar results.

Efflux of cholesterol to HDL2 from epithelioid SMCs also was reduced, by ≈40%; however, this rose to the same level as in spindle SMCs in the presence of an acyl CoA:cholesterol acyltransferase inhibitor (Figure VII in the online-only Data Supplement). This suggests that cholesterol can be mobilized to a pool available for HDL-dependent, but not apoA-I–dependent, efflux in epithelioid SMCs.

Transfection of SMCs with full-length ABCA1 cDNA to specifically upregulate ABCA1 expression similarly resulted in increased total and cell surface ABCA1 in spindle and epithelioid cells (Figure 8A). Protein kinase A–dependent phosphorylation of ABCA1, a determinant of constitutive ABCA1 activity,30 was also increased in transfected cells of both phenotypes. Despite appropriate cellular localization and phosphorylation, however, increased ABCA1 again failed to increase cholesterol efflux to apoA-I from epithelioid cells compared with the large increase seen in spindle SMCs (Figure 8B). In contrast to previous reports indicating increased apoA-I binding after transfection of ABCA1 in macrophages and HEK293 cells26,31 and despite increased cholesterol efflux in spindle SMCs, increased ABCA1 expression after transfection resulted in no increase in apoA-I binding to either spindle or epithelioid SMCs (Figure 8C).

Figure 8. Cell surface localization of ABCA1, cholesterol efflux, and binding of apoA-I to arterial SMCs transfected with ABCA1 cDNA. A, SMCs were grown to 60% to 70% confluence and then transfected with ABCA1 cDNA (ABCA1) or empty vector (mock) for 24 hours in the absence of excess cholesterol loading. Cell surface proteins were then biotinylated, and levels of total ABCA1, cell surface ABCA1, ABCA1 phosphorylated by PKA (phospho-ABCA1), and protein disulfide isomerase (PDI; loading control) were determined by Western blotting as described in Methods. B, SMCs at 60% to 70% confluence were labeled with [3H]cholesterol for 24 hours (up to ≈90% confluence), transfected with ABCA1 or empty vector for 24 hours (cells then at full confluence), and incubated with 10 μg/mL apoA-I for 4 hours. At the end of the incubation period, media and cells were collected separately, and cells were homogenized. Media and aliquots of cell homogenates were analyzed for [3H]cholesterol, and 100 μg cell protein was used to detect total ABCA1 levels by Western blotting. Cholesterol efflux data are expressed as percentage of total cell plus medium [3H]cholesterol in the medium and are mean±SD of quadruplicate determinations. Results are representative of 2 experiments with similar results. C, SMCs at 60% to 70% confluence were transfected with ABCA1 construct or empty vector for 24 hours and then incubated for 2 hours at 4°C with 10 μg/mL 125I-apoA-I ±200 μg/mL unlabeled apoA-I. Cells were then rinsed extensively and assessed for radioactivity. Cell surface 125I–apoA-I bound per 1 mg cell protein was determined by subtracting values in the presence of unlabeled apoA-I. Results are the mean±SD of quadruplicate determinations. Parallel dishes transfected with ABCA1 or empty vector but without the addition of 125I-apoA-I were harvested, and the expression levels of crude membrane ABCA1 were determined by Western blotting. Results are representative of 3 experiments with similar results.

It is possible that despite the increase in total, cell surface, and phosphorylated ABCA1, ABCA1 remains inactive in epithelioid SMCs. Transfection with ABCA1 resulted in an increase in cholesterol oxidase–sensitive cholesterol in both spindle and epithelioid SMCs, suggesting that ABCA1 increased the mobilization of cholesterol to the outer leaflet of the plasma membrane in both cell types (Figure VIIIA of the online-only Data Supplement). Hydrolysis of cholesteryl esters was intact in epithelioid SMCs, ruling out impairment of neutral cholesteryl ester hydrolase as a reason for impaired cholesterol efflux by these cells (Figure VIIIB in the online-only Data Supplement).

Discussion

In this study, we demonstrate that ABCA1 expression, apoA-I binding, and HDL particle formation are impaired in model intimal SMCs but intact in media-source arterial SMCs in culture and that ABCA1 expression is reduced in human atherosclerotic intimal- compared with medial-layer SMCs. Remarkably, correction of ABCA1 expression with an LXR agonist or transfection with full-length ABCA1 cDNA failed to correct apoA-I binding and apoA-I–mediated lipid efflux in cultured epithelioid SMCs. These results provide the first evidence of impaired ABCA1 expression in intimal SMCs and a likely reason for the overaccumulation of cholesterol in at least a subset of intimal smooth muscle foam cells. They also suggest that, in addition to reduced ABCA1 expression, some intimal SMCs may lack an additional factor or factors required for apoA-I to bind to cells and acquire lipids made available by the membrane transport activity of ABCA1 to form HDL.

Impaired ABCA1 expression and activity were determined in cultured epithelioid SMCs on the basis of markedly reduced ABCA1 mRNA and protein levels, the near absence of efflux of radiolabeled phospholipids and cholesterol and cholesterol mass to apoA-I, and an inability of these cells to form HDL particles with increased lipid content or α-mobility on 2-dimensional gels. The ability of cells to generate α- but not pre-β–mobility HDL has previously been shown to require ABCA1 activity.32,33 Consistent with low ABCA1 expression in cultured intima-type SMCs, immunohistochemistry of human atherosclerotic coronary arteries demonstrated reduced ABCA1 expression specifically in arterial intimal SMCs. To correct for the reduced cell density and known reduction of smooth muscle α-actin expression in intimal SMCs (Figure 4A),15 we normalized the colocalization of smooth muscle α-actin and ABCA1 to total smooth muscle α-actin–staining area in the intima and media. This analysis reveals a significant reduction of total intimal compared with medial SMC ABCA1 expression, despite the finding that deep intimal SMCs show relatively high ABCA1 expression compared with more proximal intimal SMC. Previous studies have reported both increased levels of ABCA1 mRNA11,12 and lower ABCA1 protein levels11,13 in whole atherosclerotic arterial extracts but did not determine ABCA1 mRNA or protein in specific arterial layers. Our results are the first demonstration of a reduction of intimal- compared with medial-layer ABCA1 mRNA and protein, specifically intimal SMC ABCA1 expression. In addition to reduced ABCA1 mRNA in intimal compared with medial layers, we cannot rule out the possibility that reduced ABCA1 stability also contributes to the lower ABCA1 protein in the intima and intimal-layer SMCs. Rong and colleagues6 have previously reported conversion of SMCs taken from the medial layer of adult mouse aorta to a macrophage-like state on cholesterol loading and ABCA1 expression by these cells. Although different subtypes of SMCs likely exist in the intima, our results indicate that impaired ABCA1 expression would explain the excess cholesterol accumulation in at least a subset of intimal SMCs.

The reduced expression of ABCA1 in rat epithelioid SMCs, also found in cultured human epithelioid SMCs (Figures III and IV in the online-only Data Supplement), was increased to levels seen in unstimulated spindle SMCs by agonists of nuclear receptors LXR and retinoid X receptor. Despite appropriate localization of the increased ABCA1 to the cell surface, this treatment failed to increase lipid efflux to apoA-I or HDL particle formation and increased the ability of apoA-I to be cross linked to ABCA1 only slightly. Similarly, transfection of full-length ABCA1, resulting in increased total, cell surface, and phosphorylated ABCA1, failed to increase both binding and cholesterol efflux to apoA-I by epithelioid SMCs. This result was seen despite evidence of activity of the transfected ABCA1 in increasing mobilization of cholesterol to a cholesterol oxidase–sensitive pool in both cell types.

Therefore, reduced ABCA1 expression in model intimal SMCs does not appear to explain the reduced apoA-I binding to these cells. Correction of ABCA1 expression, localization, and apparent activity did not alter the low levels of apoA-I binding to these cells. These findings suggest that in addition to impaired ABCA1 expression, epithelioid SMCs lack a factor or factors, possibly a cell surface or cytoskeletal component, required for apoA-I to bind to cells and receive lipids made available for HDL formation by ABCA1. The striking absence of apoA-I binding in epithelioid SMCs compared with spindle SMCs despite normalization of ABCA1 expression provides an excellent model to study the nature of this additional binding factor or factors.

Conclusions

We have demonstrated that spindle-morphology, media-phenotype SMCs exhibit high levels of ABCA1 expression, apoA-I binding, and efflux of cellular lipids to form HDL particles, whereas intima-phenotype SMCs lack these features. We have also shown that correction of ABCA1 expression fails to correct apoA-I binding or HDL production by intima-type cells. Human atherosclerotic intimal SMCs similarly exhibit impaired ABCA1 expression, suggesting a novel explanation for the development of intimal smooth muscle foam cells in vivo.

Acknowledgments

This work was funded by a Heart and Stroke Foundation of Alberta, Northwest Territories, and Nunavut grant-in-aid, CIHR operating grant MOP-12660, an unrestricted research grant from Pfizer Canada to Dr Francis, CIHR FRN MOP-49589 to Dr McManus, and CIHR FRN MOP-11715 to Dr Pickering. Drs Choi and Rahmani were recipients of the Heart and Stroke Foundation of Canada Doctoral Research Awards. B.W. Wong was supported by a CIHR Doctoral Research Award and a Michael Smith Foundation for Health Research Senior Graduate Studentship. Dr Pickering is a career investigator of the Heart and Stroke Foundation of Ontario. Dr Francis was a senior scholar of the Alberta Heritage Foundation for Medical Research.

CLINICAL PERSPECTIVE

Arterial smooth muscle cells (SMCs) are a major cellular component of atherosclerotic lesions and, like macrophages, accumulate excess cholesterol. This study provides evidence that expression of the major mediator of cholesterol removal from cells and the rate-limiting protein in new high-density lipoprotein particle formation, ATP-binding cassette transporter A1 (ABCA1), is reduced in intima-type cultured SMCs, in human coronary atherosclerotic intima, and specifically in atherosclerotic intimal SMCs. Remarkably, overexpression of ABCA1 in cultured intima-type SMCs failed to correct the binding of the main protein of high-density lipoprotein, apolipoprotein A-I, to the cells or cholesterol efflux from these cells. These results provide a previously unknown explanation for the accumulation of excess cholesterol in intimal smooth muscle foam cells and suggest that differences in gene and protein expression by medial and intimal SMCs might identify apolipoprotein A-I binding factors required for new high-density lipoprotein particle formation. Identification of these factors would provide novel targets for raising high-density lipoprotein clinically for the prevention of atherosclerosis.

Footnotes

The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.841130/DC1.